Large-scale cortical reorganization following forelimb deafferentation in rat does not involve plasticity of intracortical connections.

Physiological mapping of the body representation 1 month or longer after forelimb removal in adult rats revealed new pockets of shoulder representation in the forepaw barrel subfield (FBS) in the first somatosensory cortex (SI). These "new" shoulder representations have longer evoked response latencies than sites in the shoulder representation within the trunk subfield, hereafter referred to as the "original" shoulder representation. We postulated that the "new" shoulder representations in the FBS were relayed from the "original" shoulder representation. We investigated this hypothesis by studying anatomical connectivity between the "original" shoulder representation and the FBS in intact control and forelimb deafferented adult rats using Phaseolus vulgaris leucoagglutinin (PHA-L), biocytin, and biotin dextran-amine (BDA) as anterograde tracers. The retrograde tracer cholera toxin beta subunit (CT-B) injected into the FBS was also used to study connectivity between the "original" shoulder representation and the FBS. Using these anatomical tracing techniques, we were unable to show the existence of a direct corticocortical connection between the "original" shoulder representation in the trunk subfield and the FBS in either intact or deafferented rats. Functional connectivity between the two cortical regions was studied by ablating the "original" shoulder representation alone or in combination with the shoulder representation in the second somatosensory cortex (SII) while recording evoked responses in the FBS following electrical stimulation of the shoulder. Both ablations failed to eliminate the evoked responses at the "new" shoulder sites in the FBS, suggesting that SI and SII are not necessary for "new" shoulder input in the FBS. It is suggested that subcortical sites may play a major role in large-scale cortical reorganization. Results of projections from the "original" shoulder representation to parietal medial (PM), parietal lateral (PL), SII, parietal ventral (PV), and parietal rhinal (PR) sensory fields and agranular lateral (AgL) and agranular medial (AgM) motor fields are also described.

Location and distribution of Fos protein expression in rat hippocampus following acute moderate aerobic exercise.

Hippocampal neurons are activated during endurance exercise; however, little attention has been given to the location and spatial distribution of these neurons. We have, therefore, used Fos protein expression to identify the location and distribution of hippocampal neurons that become activated during acute moderate aerobic exercise. Adult rats were assigned into trained running (TR), trained nonrunning (TNR), untrained nonrunning (UNR), and cage-bound (CB) groups. Rats in the TR and TNR groups were trained to run, for three 20-min running periods separated by 3 min rest, on a treadmill. Rats in the UNR group spent identical time on a nonactivated treadmill, while rats in the CB group remained in their home cages throughout the training and experimentation. After training to criterion performance for both TR and TNR groups, both groups were rested for 1 day. Rats in the TR were then run on the treadmill to criterion level, while those in TNR and UNR groups spent equivalent time on the nonactivated treadmill. Animals in all groups were then killed and their brains removed, sectioned, and processed for Fos protein immunocytochemistry. Fos-like immunoreactive (FLI) neurons were counted in the dentate and CA1-3 fields of the hippocampus. The total numbers of hippocampal FLI neurons, as well as FLI neurons in each hippocampal region, were compared among groups. The total numbers of FLI neurons in the hippocampus, as well as in individual regions, were significantly greater in the TR group compared with the other three groups. Similarly, significant differences were found between the TNR group when compared with UNR and CB groups. Conversely, a significant difference existed between UNR and CB only in the CA1 field, which may account for the significant difference in the total number of hippocampal FLI neurons between these two groups. These results show that Fos induction occurs in the hippocampus during moderate physical exercise. Furthermore, the importance of the incorporation of adequate controls to account for possible differences in expression of immediate early gene expression due to trained performing, trained nonperforming, and untrained groups is discussed. The results indicate that adequate control for nonexercise stimuli is necessary for studies of the effect of exercise on the brain when expression of immediate early genes such as c-fos is used as an outcome measure.

Thalamocortical arbors extend beyond single cortical barrels: an in vivo intracellular tracing study in rat.

Neurons in layer IV of rat somatosensory (SI) barrel cortex receive punctate somatic input from well-defined regions of the periphery. Following peripheral deafferentation, SI neurons in deafferented cortex respond to new input from neighboring regions of the skin surface. The precise mechanism(s) through which this occurs is unknown, although corticocortical and barreloid to barrel connections have been suggested as possible substrates. Because layer-IV barrels receive a strong afferent input from ventroposterior (VP) thalamic projection neurons, any divergence in the thalamocortical (TC) projection to multiple cortical barrels could also provide an anatomical substrate for rapid cortical reorganization. We used in-vivo intracellular recording methods to record and physiologically identify neurons in rat VP and to label those neurons with an intracellular tracer. Thalamic neurons (n=117) were impaled with sharp intracellular electrodes, and the receptive field(s) and firing pattern were measured. Cells were then injected with biocytin or biotinylated dextran amine (BDA). A total of 38 labeled TC neurons were quantitatively analyzed for soma size and dendritic arborization size; quantitative analysis of TC-axon arborizations in layer IV of barrel cortex was carried out in a total of 13 TC neurons. Two different axon-arborization patterns were identified in SI cortex: direct-projecting axons (n=6) were observed to project to and arborize within a single cortical barrel as well as extend their fibers into adjacent barrels; bifurcating-type axons (n=7) were seen to bifurcate in the subcortical white matter or in layer VI and then project to multiple barrel columns, where they arborized in layer IV. Axon fibers were always observed in three or more cortical barrels (mean=5, range=3-7). The mean mediolateral extent of arborizations in layer IV for the direct-projecting and bifurcating type axons were 458 microm and 1,302 microm, respectively, and these were significantly different (t=3.78, P<0.01). Axon-fiber length within cortical laminae was measured for each arborization pattern in relationship to the total fiber length within a cortical column. Direct-projecting axons always had greater than 50% of their fiber length within layer IV. Bifurcating-type axons were differentially distributed within multiple columns and always had less than 50% of their total column fiber length in layer IV. Morphological analysis of TC somata and dendrites revealed no correlation between local neuron morphology and axonal-arborization patterns. All intracellularly recorded TC neurons had similar adapting firing patterns when injected with a long-duration pulse. Our results showed that TC neurons project to multiple cortical barrels with one barrel receiving the principal input. This divergent TC projection pattern in SI cortex may provide an anatomical substrate for cortical plasticity and must be considered in any mechanism of rapid cortical reorganization.

EDTA chelation effects on urinary losses of cadmium, calcium, chromium, cobalt, copper, lead, magnesium, and zinc.

The efficacy of a chelating agent in binding a given metal in a biological system depends on the binding constants of the chelator for the particular metals in the system, the concentration of the metals, and the presence and concentrations of other ligands competing for the metals in question. In this study, we make a comparison of the in vitro binding constants for the chelator, ethylenediaminetetraacetic acid, with the quantitative urinary excretion of the metals measured before and after EDTA infusion in 16 patients. There were significant increases in lead, zinc, cadmium, and calcium, and these increases roughly corresponded to the expected relative increases predicted by the EDTA-metal-binding constants as measured in vitro. There were no significant increases in urinary cobalt, chromium, or copper as a result of EDTA infusion. The actual increase in cobalt could be entirely attributed to the cobalt content of the cyanocobalamin that was added to the infusion. Although copper did increase in the post-EDTA specimens, the increase was not statistically significant. In the case of magnesium, there was a net retention of approximately 85% following chelation. These data demonstrate that EDTA chelation therapy results in significantly increased urinary losses of lead, zinc, cadmium, and calcium following EDTA chelation therapy. There were no significant changes in cobalt, chromium, or copper and a retention of magnesium. These effects are likely to have significant effects on nutrient concentrations and interactions and partially explain the clinical improvements seen in patients undergoing EDTA chelation therapy.

Effects of large-scale limb deafferentation on the morphological and physiological organization of the forepaw barrel subfield (FBS) in somatosensory cortex (SI) in adult and neonatal rats.

The physiological representation of the shoulder and surrounding body was examined in layer IV of somatosensory cortex (SI) in rats that had underground removal of the forelimb, either as newborns on postnatal day three (PND-3) or as adults (at least 8 weeks of age). Electrophysiological recordings were used to map the shoulder and body representations (physiological map), and the mitochondria marker, cytochrome oxidase (CO), was used to visualize recording sites in barrel and barrel-like structures (morphological map) in layer IV of deafferents and intact controls. The SI shoulder representation lies in a nebulously stained region that lies posterior to the forearm, wrist, and forepaw representations; the latter region is associated with the well-defined forepaw barrel subfield (FBS). The major findings are: (1) the shoulder is represented as a single zone located at the posterior extent of the SI body map in intact rats; (2) limb deafferentation in adult or neonatal rats that were physiologically mapped 6-16 weeks post-amputation resulted in two or more islets of "new" representation of the shoulder in the FBS in addition to the representation of the "original" shoulder in the posterior part of the body map; (3) deafferentations made in neonatal rats, physiologically mapped as adults, had a significantly greater (Mann-Whitney U) amount of "new" cortical representation within the FBS than did rats deafferented as adults; (4) fewer unresponsive sites in the FBS were found for neonate deafferents than for adult deafferents; (5) evoked response latencies following electrical stimulation of the shoulder were shortest for cortical sites within the "original" shoulder representation in intact controls, and latencies recorded at the "original" shoulder representation in deafferents were also shorter than latencies recorded in "new" shoulder representations in both groups of deafferents; and (6) morphological maps of the FBS were altered in neonate deafferents to the extent that the barrel structure was poorly formed, as exemplified by the absence of the four mediolateral running bands; however, the overall ovoid shape of the FBS was still apparent, but not as sharply defined as for intact controls or adult deafferents. Possible mechanisms for reorganization following large-scale deafferentation are discussed.

Specificity in the interaction of HVA Ca2+ channel types with Ca2+-dependent AHPs and firing behavior in neocortical pyramidal neurons.

Intracellular recordings and organic and inorganic Ca2+ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca2+ channels are differentially coupled to Ca2+-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca2+ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N-type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between approximately 80 and 90% of the slow AHP (sAHP) were Ca2+ dependent. Based on the effects of specific organic Ca2+ channel blockers (dihydropyridines, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca2+ current subtypes.

In vivo intracellular recording and labeling of neurons in the forepaw barrel subfield (FBS) of rat somatosensory cortex: possible physiological and morphological substrates for reorganization.

We examined the physiological properties and morphology of neurons in the forepaw barrel subfield (FBS) in somatosensory cortex (SI) of adult rats using in vivo intracellular recording and biocytin labeling techniques. Our results show that both pyramidal and non-pyramidal type spiny neurons can be activated with short latency by peripheral stimulation. FBS neurons within individual barrels receive both suprathreshold and subthreshold convergent input from one or more forepaw digits and pads. We hypothesize that some of these subthreshold inputs may be elevated to firing level by some, as yet unknown, mechanism(s) following peripheral deafferentation. Examination of the relationship between the dendritic pattern of labeled neurons and individual barrels within the FBS suggests that neurons with dendrites extending into neighboring barrel bands may serve as a possible morphological substrate for immediate reorganization.

Relationship between representation of hindpaw and hindpaw barrel subfield (HBS) in layer IV of rat somatosensory cortex.

We describe the organization of the hindpaw barrel subfield (HBS) in layer IV of rat somatosensory cortex (SI) and relate this organization to the representation of the hindpaw. The ovoid-shaped, HBS is oriented anterior to posterior and comprises barrels and barrel-like structures, the most prominent of which consist of at least five anteriorly-located elongated barrel bands. Posterior to these elongated bands is a cluster of four barrels. Two additional barrels are found, one lateral, the other medial. The lateral border is formed by a nearly continuous band that overlaps portions of the anterior elongated bands and posterior barrels. The HBS shows considerable variability in size and shape; nevertheless, the overall pattern reflects a common plan of organization. Electrophysiological mapping confirmed that hindpaw representation is somatotopically organized. The glabrous toes are represented anteriorly, the pads posteriorly, and the dorsal hairy skin of the toes and hindpaw laterally. By aligning physiological and morphological (HBS) maps according to lesion sites, our data suggest that the elongated anteriorly-located barrel bands represent the hindpaw toes, the four toe pads are represented immediately posterior followed by barrels representing the plantar pads. The representations of dorsal hairy skin of toe and dorsal hindpaw form the lateral border; the heel and ankle are represented most posterior. We interpret our findings as support that individual barrels in the HBS are associated with discrete regions of the hindpaw; however, the precise relationship of structure and function reported between the vibrissae and posteromedial barrel subfield (PMBSF) and between the forepaw and the forepaw barrel subfield (FBS) were not observed.

Electrical stimulation of a forepaw digit increases the physiological representation of that digit in layer IV of SI cortex in rat.

We studied the physiological representation of digit three (D3) in rat somatosensory cortex (SI) before and immediately after electrical stimulation (1.5x threshold for 2 h) of the glabrous tip of D3 in anesthetized animals (n = 6). Measurements of D3 representation were also made in anesthetized non-stimulated control animals (n = 2). The post-stimulation areal measurements of D3 representation in experimental animals were statistically significantly larger than both pre-stimulation measurements in experimental animals and post-stimulation measurements in control animals. Our results suggest that short-term electrical stimulation is sufficient to expand the D3 representation in each of the experimental animals, while the maps in non-stimulated controls showed little variation. The fact that these studies were carried out in anesthetized animals suggests that the results are independent of the state of the animal. The present findings emphasize the importance of afferent input in modulating cortical organization.

Digit removal leads to discrepancies between the structural and functional organization of the forepaw barrel subfield in layer IV of rat primary somatosensory cortex.

The physiological representation of the forepaw in rat primary somatosensory cortex (SI) is topographically organized. This representation is associated with the unique arrangement of barrels in layer IV of the forepaw barrel subfield (FBS) in SI and provides an example of a relationship between cortical structure and function. It has been reported that removal of peripheral afferent input to the FBS prior to postnatal day 5 or 6 results in a disorganized FBS, while deafferentation at later times produces little or no alteration of the FBS. Therefore, restricted deafferentations of individual digits in adult rats should result in little, if any, disruption of the FBS, while at the same time eliminating afferent input to the FBS from a localized region of the periphery. This manipulation is likely to create a mismatch between structure and function and offer insight into what barrels actually represent in the adult deafferent. In the present study, we amputated digit three (D3) in eight adult rats, allowed a 1-month survival time, physiologically mapped the representation of D2, D4, and the stump, and compared this physiological map to the underlying barrels in the FBS. Our results showed that FBS barrels formerly associated with the representation of D3 were now associated with the representation of surrounding digits D2 and D4, as well as the remaining stump. By superimposing the morphological and physiological map upon one another, it was clear that the D2 and D4 representations expanded into the former D3 barrel territory and septae between the barrels. The reorganized physiological map was somatotopically organized, even though the general configuration of the morphological map remained unaltered, as visualized with cytochrome oxidase staining. These results suggest that in the deafferent, neurons within FBS barrels previously associated with the representation of punctate regions of skin become associated with neighboring regions of skin. A morphological substrate to account for this cortical reorganization is described.

Relationship between the organization of the forepaw barrel subfield and the representation of the forepaw in layer IV of rat somatosensory cortex.

We studied the organization of the forepaw barrel subfield (FBS) in layer IV of adult rat somatosensory cortex using the mitochondrial marker cytochrome oxidase and related this organization to the representation of the forepaw. The FBS is an ovoid structure consisting of barrels and barrel-like structures, the most conspicuous of which form four centrally located medio lateral running bands. Each band contains three to four barrels. These centrally located bands are bordered along their entire lateral side by a nebulous zone of undifferentiated labeling. At the anterior border, two small barrels are located laterally and one or two larger barrels are located medially. Medial to the central zone are three well-defined barrels. The posterior border consists of a nebulous field of labeling and occasional barrel-like structures. The results from our electrophysiological recording and mapping revealed that the forepaw representation was topographically organized into a single map and that the forepaw map matches almost precisely with individual barrels and barrel-like structures in the FBS. Each of the four central bands is associated with the representation of a single glabrous digit. Digit two (D2) is represented anteriorly and followed posteriorly by D3 through D5. Within each digit band the digit is somatotopically organized, with the skin over the distal phalanx represented in the two lateral barrels and the middle and proximal phalanges represented in the medial barrel(s). The dorsal hairy digit skin and dorsal hand are represented in the lateral zone. D1 is represented by two small anteriorly located barrels. Medial to the representation of the glabrous digits is the representation of the palmar pads. The representation of these pads, in turn, lies between the representations of the thenar (located anteriorly) and hypothenar (located posteriorly) pads. Posterior to the hypothenar pad representation lie the representations of the wrist and forearm. While the present results support the conclusion that individual barrels are associated with discrete locations on the forepaw, examples were found where the recording site was not precisely located within the predicted barrel. Some of these errors may be accounted for by limitations in the mapping techniques; nevertheless, the FBS offers an excellent model system to study relationships between cortical structure and function.

Large unresponsive zones appear in cat somatosensory cortex immediately after ulnar nerve cut.

The organization of the primary somatosensory cortex innervated by the ulnar nerve was studied before and immediately after ulnar nerve transection in 11 cats electrophysiologically mapped under Nembutal or Ketamine anesthesia. The cortex was reexamined a second time beginning 42 hr after nerve transection in four cats anesthetized with Nembutal. One additional sham-operated control was also mapped. The region of cortex formerly served by the ulnar nerve remained largely unresponsive to somatic stimulation independent of the type of anesthetic used during recording. Nonetheless, animals anesthetized with Ketamine had more new responsive sites in deafferented cortex following nerve cut than cats anesthetized with Nembutal. New responses, when observed, were evoked by stimulation of a region of skin adjacent to the region served by the ulnar nerve. These findings suggest that the immediate response to deafferentation of somatosensory cortex is a limited acquisition of novel responses restricted to a region immediately adjacent to cortex containing normal afferent input.

Reevaluation of area 3b in the cat based on architectonic and electrophysiological studies: regional variability with functional and anatomical consistencies.

Most anatomical and electrophysiological studies of the cat primary somatosensory cortex rely on Hassler and Muhs-Clement's (J. Hirnforsch. 6:377-420, 1964) cyto- and myeloarchitectonic description distinguishing area 3a from area 3b; however, discrepancies in the delineation of these areas in published studies suggest that many workers have found it difficult to apply those criteria systematically. We examined the cytoarchitecture of area 3b in Nissl stained sagittal sections from which electrophysiological data had been obtained prior to sacrifice. Rostrocaudal rows of electrode penetrations placed at different mediolateral positions in the gyrus located regions responsive to stimulation of either cutaneous or deep structures. Small electrolytic lesions allowed these data to be related to the cytoarchitecture. A systematic study throughout the trunk and limb representations found cutaneous responses in cortical regions characterized by a thick and cell-dense granular layer IV, however these same regions had a variable population of medium-sized and/or large pyramidal cells in layer V. Pyramidal cells were practically absent from the forelimb representation, but were present to varying degrees in the trunk and hindlimb representations. Moreover, the relative thickness and cell-density in layer IV were greater in the forelimb than in the hindlimb representations. Deep responses were found in cortex characterized by a thinner layer IV. Since the characteristics of layer V in area 3a were variable, it was less useful for identification of the border between areas 3a and 3b. Clear changes in the intensity and laminar distribution of acetylcholinesterase staining occurred between areas 3a and 3b, making this a useful adjunct to the Nissl stain.

Early development of the SI cortical barrel field representation in neonatal rats follows a lateral-to-medial gradient: an electrophysiological study.

Development of the barrel field in layer IV of SI cortex of neonatal rats was studied in vivo using electrophysiological recording techniques. This study was designed to determine (a) the earliest time SI cortex is responsive to peripheral mechanical and/or electrical stimulation and (b) whether the development of the SI cortical barrel field map of the body surface follows a differential pattern of development similar to the pattern previously demonstrated using peanut agglutinin (PNA) binding (McCandlish et al. 1989). Carbon fiber microelectrodes were used to record evoked responses from within the depth of the cortex in neonatal rats between postnatal day 1. (PND-1), defined as the day of birth, and PND-14. Evoked responses were first recorded approximately 12 h after birth. These responses in the youngest animals were of low amplitude, monophasic waveshape, and long latency, with long interstimulus intervals necessary to drive the cortex. Increases in amplitude and complexity of waveshape and decreases in latency were observed over subsequent postnatal days. The earliest responses recorded on middle PND-1 were evoked by stimulation of the face and/or mystacial vibrissae. The next responses were evoked approximately 24 h after birth (late PND-1) by stimulation of the forelimb. The last responses were evoked approximately 36 h after birth (middle PND-2), by stimulation of the hindlimb. The physiological map of the representation of the body surface follows a developmental gradient similar to the gradient observed using PNA histochemistry; however, the lectin-generated morphological map lagged approximately 48 h behind the physiological map.(ABSTRACT TRUNCATED AT 250 WORDS)

Contributions of low-threshold calcium current and anomalous rectifier (Ih) to slow depolarizations underlying burst firing in human neocortical neurons in vitro.

The slow depolarization that underlies the voltage-dependent burst-firing behavior of human neocortical neurons is mediated by a low-threshold calcium conductance in concert with the anomalous rectifier current, Ih. The slow depolarization could be elicited by depolarization from negative membrane potentials or as a rebound following hyperpolarization. The rebound depolarization was time- and voltage-dependent. Most of the slow depolarization was blocked by inorganic calcium blockers. The remainder of the depolarization and the 'sag' in the hyperpolarizing voltage responses were blocked by extracellular Cs+.

Organization of the mouse motor cortex studied by retrograde tracing and intracortical microstimulation (ICMS) mapping.

The motor representation of the body musculature was studied in 11 adult mice by using ICMS. The motor responses elicited from both granular and agranular cortical fields showed that the mouse motor cortex is topographically organized; however, within the representation of individual body-parts the movements are multiply represented. In addition, several sites were encountered where more than one movement was elicited at the same stimulus threshold. The locations of pyramidal cells contributing axons to the pyramidal tract were examined by means of retrograde tracing with HRP injected into the cervical enlargement. This procedure labeled neurons only in lamina V in granular and agranular cortical fields. The similarities between the organization of motor cortex demonstrated in this study and the organization in the rat suggest that the rat and mouse share a common plan of rodent motor cortical organization.

Ulnar nerve innervation of paw and SI cortex of cat: substrate for reorganization.

We studied the distribution of the peripheral nerves innervating the distal forepaw by recording receptive fields from fascicles of the ulnar, radial, and median nerves and compared this result with the peripheral nerve representation in primary somatosensory (SI) cortex of cat. Our findings suggest that SI cortex receives input, in large part, from multiple peripheral nerves even when those nerves do not show a strong overlapping pattern in the periphery. This overlap pattern observed in SI cortex may be responsible, in part, for the immediate reorganization which is known to follow peripheral nerve deafferentation.